Cancer can be considered a disease of genetic instability. DNA damage triggers a complex response that includes cell cycle arrest and coordinated activation of DNA repair. Failure to activate or to coordinate the DNA-damage induced signal transduction pathways can lead to chromosome breakage and loss, and to the propagation of mutations. Indeed, several cancer-prone syndromes reflect defects in the DNA damage response and are characterized by genetic instability. These include, but are not limited to, Ataxia- Telangiectasia, Nijmegen Breakage Syndrome, Ataxia-Telangiectasia Like Disorder, Li-Fraumeni Syndrome and familial forms of breast and cervical cancers. Our long-term objective is to understand the molecular nature of the signal transduction pathways activated following DNA damage. To that goal, we have established unique cell-free systems derived from Xenopus eggs that faithfully recapitulate several aspects of the DNA damage response. We will use these cell-free systems to screen for proteins that are modified in presence of double strand breaks (DSBs). Next, we will investigate the role in the DNA damage response of a candidate gene we have identified in a pilot screen and of additional genes we will isolate. We will screen a chemical library for compounds that interfere with the in vitro activation of ATM/ATR protein kinases, an early step of the DNA damage response. The compounds identified will be further validated and their activity will be tested in a variety of assays performed in Xenopus cell-free systems. We anticipate that these studies will help identify novel components of the biochemical pathways activated by DNA damage and shed light on the nature of the biochemical steps constituting these pathways. These studies will provide valuable information on how the DNA damage response can be impaired or lost in the case of cancer and will help identify drugs that influence the DNA damage response.